In Vitro DNA Binding




A SAGE like SELEX protocol is being used to determine the in vitro DNA binding specificities of transcription factors. We currently have data for 11 of the 37 principal regulators of the early embryo network. Our first public release of data will be in late 06. Transcription factor proteins tagged with six-his are first immobilized on metal affinity resins and then used to select DNAs from a mixed sequence pool of oligonucleotides. The process is repeated using the amplified pool of selected oligonucleotides from the previous round as input DNA to the next round of selection. The sequences of around 1,000 selected oligonucleotides are determined for each successive round, capturing many binding site variants. An automated software pipeline is then used derive Position Weight Matrices (PWMs) for each round of SELEX. The sequences of oligonucleotides from each round of SELEX and derived PWMs will be made available.

We have also developed a multiplex method that can measure DNA binding of resin immobilized protein to 15 different oligonucleotides at once and have used it to measure the relative affinities of binding site variants for several factors.

Our in vitro DNA binding data has been invaluable for determining the locations of recognition sites found in intervals bound in ChIP/chip experiments and for subsequent comparative analyses.